YAP1 regulation by cell density independent of N-cad. (A) Effect of N-cad depletion on YAP1 localization in migrating PBT-05 cells. (B and C) Effect of extracellular N-cad on YAP1 localization. Representative images of cells migrating on 12.5 μg/ml N-cad ECD-Fc coated surface for 24 h and quantification of percent nuclear YAP1 after migration on 6.25, 12.5, or 25 μg/ml N-cad ECD-Fc. (D) Representative images of control and N-cad shRNA cells on 40 or 70 μm laminin-coated discs for 48 h before fixation and staining for YAP1 and N-cad. The number of cells per cluster is noted in the lower left. Arrowheads indicate cells with nuclear YAP1. Scale bar, 20 μm. (E) Quantification of percent cells with nuclear YAP1 in different size clusters of control and N-cad shRNA cells. (F) N-cad antibody uptake by cells in different size clusters. (A and C) Two-way ANOVA Šídák’s multiple comparisons test. Error bars show mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.001, ****P < 0.0001. (A)N = 6–7 spheroids, three experiments. (B)N = 3 spheroids in each condition. (E and F) The number of clusters quantified is noted on the graph.